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- All Subjects: Cellular Biology
- Genre: Academic theses
- Creators: Blattman, Joseph N
- Creators: Kusumi, Kenro
- Member of: ASU Electronic Theses and Dissertations
- Resource Type: Text
Description
Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) is a rare and highly aggressive ovarian cancer that affects children and young women at a mean age of 24 years. Most SCCOHT patients are diagnosed at an advanced stage and do not respond to chemotherapy. As a result, more than 75% of patients succumb to their disease within 1-2 years. To provide insights into the biological, diagnostic, and therapeutic vulnerabilities of this deadly cancer, a comprehensive characterization of 22 SCCOHT cases and 2 SCCOHT cell lines using microarray and next-generation sequencing technologies was performed. Following histological examination, tumor DNA and RNA were extracted and used for array comparative genomic hybridization and gene expression microarray analyses. In agreement with previous reports, SCCOHT presented consistently diploid profiles with few copy number aberrations. Gene expression analysis showed SCCOHT tumors have a unique gene expression profile unlike that of most common epithelial ovarian carcinomas. Dysregulated cell cycle control, DNA repair, DNA damage-response, nucleosome assembly, neurogenesis and nervous system development were all characteristic of SCCOHT tumors. Sequencing of DNA from SCCOHT patients and cell lines revealed germline and somatic inactivating mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 79% (19/24) of SCCOHT patients in addition to SMARCA4 protein loss in 84% (16/19) of SCCOHT tumors, but in only 0.4% (2/485) of other primary ovarian tumors. Ongoing studies are now focusing on identifying treatments for SCCOHT based on therapeutic vulnerabilities conferred by ubiquitous inactivating mutations in SMARCA4 in addition to gene and protein expression data. Our characterization of the molecular landscape of SCCOHT and the breakthrough identification of inactivating SMARCA4 mutations in almost all cases of SCCOHT offers the first significant insight into the molecular pathogenesis of this disease. The loss of SMARCA4 protein is a highly sensitive and specific marker of the disease, highlighting its potential role as a diagnostic marker, and offers the opportunity for genetic testing of family members at risk. Outstanding questions remain about the role of SMARCA4 loss in the biology, histogenesis, diagnosis, and treatment of SCCOHT.
ContributorsRamos, Pilar (Author) / Anderson, Karen (Thesis advisor) / Trent, Jeffrey (Committee member) / Kusumi, Kenro (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2014
Description
The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is essential for the innate immune response to danger signals. Importantly, the NLRP3 inflammasome responds to structurally and functionally dissimilar stimuli. It is currently unknown how the NLRP3 inflammasome responds to such diverse triggers. This dissertation investigates the role of ion flux in regulating the NLRP3 inflammasome. Project 1 explores the relationship between potassium efflux and Syk tyrosine kinase. The results reveal that Syk activity is upstream of mitochondrial oxidative signaling and is crucial for inflammasome assembly, pro-inflammatory cytokine processing, and caspase-1-dependent pyroptotic cell death. Dynamic potassium imaging and molecular analysis revealed that Syk is downstream of, and regulated by, potassium efflux. Project 1 reveals the first identified intermediate regulator of inflammasome activity regulated by potassium efflux. Project 2 focuses on P2X7 purinergic receptor-dependent ion flux in regulating the inflammasome. Dynamic potassium imaging revealed an ATP dose-dependent efflux of potassium driven by P2X7. Surprisingly, ATP induced mitochondrial potassium mobilization, suggesting a mitochondrial detection of purinergic ion flux. ATP-induced potassium and calcium flux was found to regulate mitochondrial oxidative signaling upstream of inflammasome assembly. First-ever multiplexed imaging of potassium and calcium dynamics revealed that potassium efflux is necessary for calcium influx. These results suggest that ATP-induced potassium efflux regulates the inflammasome by calcium influx-dependent mitochondrial oxidative signaling. Project 2 defines a coordinated cation flux dependent on the efflux of potassium and upstream of mitochondrial oxidative signaling in inflammasome regulation. Lastly, this dissertation contributes two methods that will be useful for investigating inflammasome biology: an optimized pipeline for single cell transcriptional analysis, and a mouse macrophage cell line expressing a genetically encoded intracellular ATP sensor. This dissertation contributes to understanding the fundamental role of ion flux in regulation of the NLRP3 inflammasome and identifies potassium flux and Syk as potential targets to modulate inflammation.
ContributorsYaron, Jordan Robin (Author) / Meldrum, Deirdre R (Thesis advisor) / Blattman, Joseph N (Committee member) / Glenn, Honor L (Committee member) / Arizona State University (Publisher)
Created2015
Description
Malignant brain tumors are devastating despite aggressive treatments such as surgical resection, chemotherapy and radiation therapy. The average life expectancy of patients with newly diagnosed glioblastoma is approximately 15 months. One novel therapeutic strategy involves using a ketogenic diet (KD) which increases circulating ketones and reduces circulating glucose. While the preclinical work has shown that the KD increases survival, enhances radiation and alters several pathways in malignant gliomas, its impact on the anti-tumor immune response has yet to be examined. This dissertation demonstrates that mice fed the KD had increased tumor-reactive innate and adaptive immune responses, including increased cytokine production and cytolysis via tumor-reactive CD8+ T cells. Additionally, we saw that mice maintained on the KD had increased CD4 infiltration, while T regulatory cell numbers stayed consistent. Lastly, mice fed the KD had a significant reduction in immune inhibitory receptor expression as well as decreased inhibitory ligand expression on glioma cells, namely programmed death receptor -1 (PD-1) and its ligand programmed death receptor ligand -1 (PD-L1). Further, it is demonstrated that the ketone body beta-hydroxybutyrate (BHB) reduces expression of PD-L1 on glioma cells in vitro suggesting it may be responsible in part for immune-related changes elicited by the KD. Finally this dissertation also shows that the KD increases the expression of microRNAs predicted to target PD-L1 suggesting a potential mechanism to explain the ability of the KD to modulate immune inhibitory checkpoint pathways. Taken together these studies shed important light on the mechanisms underlying the KD and provide additional support for its use an adjuvant therapy for malignant glioma.
ContributorsWoolf, Eric Christopher (Author) / Compton, Carolyn C. (Thesis advisor) / Scheck, Adrienne C (Committee member) / Preul, Mark C (Committee member) / Blattman, Joseph N (Committee member) / Mehta, Shwetal (Committee member) / Arizona State University (Publisher)
Created2018
Description
Cell death is a powerful tool through which organisms can inhibit the spread of viruses by preventing their replication. In this work, I used viral and chemical stressors to elucidate the mechanisms by which one anti-viral system might be activated over another, focusing on the programmable death pathway necroptosis and Protein Kinase R (PKR). PKR can detect viral dsRNA and trigger antiviral effects such as cessation of translation and induction of programmed death. Necroptosis is a rapid cellular death that can be induced via sensors such as DNA-dependent activator of IFN-regulatory factors (DAI), also known as Z-DNA-binding protein 1 (ZBP1). DAI contains a Z-form nucleic acid (ZNA) binding domain. E3, the primary vaccinia virus (VACV) interferon resistance protein, contains a similar domain in its amino terminus. We have previously reported this domain to be necessary for the inhibition of both PKR activation and DAI/ZBP1-mediated necroptosis.
Monkeypox virus is a reemerging human pathogen. Despite a partial amino-terminal deletion in its E3 homolog, it does not activate PKR. In chapter 2, I show that MPXV produces less dsRNA than VACV, which could explain how the virus avoids activating PKR.
The amino-terminus of vaccinia is associated with ZNA binding, inhibition of PKR, and inhibition of necroptosis. To determine the roles of PKR inhibition and ZNA binding in necroptosis inhibition, I characterized the VACV mutants Za(ADAR1)-E3, which binds ZNA but does not inhibit PKR, and E3:Y48A, which cannot bind ZNA. I found that while Za(ADAR1)-E3 fails to induce necroptosis, E3:Y48A does not activate PKR but does induce necroptosis. This suggests that Z-form nucleic acid binding is not necessary for vaccinia E3-mediated inhibition of PKR, nor is the inhibition of PKR sufficient for the inhibition of necroptosis.
Finally, all known ZNA-binding proteins have immune functions and home to stress granules. I asked if stress granule formation alone could lead to necroptosis. I found that in L929 cells sodium arsenite, a known inducer of stress granules, could trigger DAI-dependent necroptosis. This suggests that DAI/ZBP1 is not necessarily a sensor of viral ligands but perhaps is a sensor of stress signals brought about by infection.
Monkeypox virus is a reemerging human pathogen. Despite a partial amino-terminal deletion in its E3 homolog, it does not activate PKR. In chapter 2, I show that MPXV produces less dsRNA than VACV, which could explain how the virus avoids activating PKR.
The amino-terminus of vaccinia is associated with ZNA binding, inhibition of PKR, and inhibition of necroptosis. To determine the roles of PKR inhibition and ZNA binding in necroptosis inhibition, I characterized the VACV mutants Za(ADAR1)-E3, which binds ZNA but does not inhibit PKR, and E3:Y48A, which cannot bind ZNA. I found that while Za(ADAR1)-E3 fails to induce necroptosis, E3:Y48A does not activate PKR but does induce necroptosis. This suggests that Z-form nucleic acid binding is not necessary for vaccinia E3-mediated inhibition of PKR, nor is the inhibition of PKR sufficient for the inhibition of necroptosis.
Finally, all known ZNA-binding proteins have immune functions and home to stress granules. I asked if stress granule formation alone could lead to necroptosis. I found that in L929 cells sodium arsenite, a known inducer of stress granules, could trigger DAI-dependent necroptosis. This suggests that DAI/ZBP1 is not necessarily a sensor of viral ligands but perhaps is a sensor of stress signals brought about by infection.
ContributorsJohnson, Brian Patrick (Author) / Jacobs, Bertram L (Thesis advisor) / Blattman, Joseph N (Committee member) / Langland, Jeffrey O (Committee member) / Stout, Valerie G (Committee member) / Arizona State University (Publisher)
Created2018
Description
Zika virus (ZIKV) outbreaks have been linked to several neurological pathologies in the developing fetus, which can progress to spontaneous abortion and microcephaly in newborns whose mothers were infected with the virus during pregnancy. ZIKV has also been correlated with neurological complications in adults such as Guillain-Barré Syndrome (GBS). ZIKV outbreaks often occur in low income areas with limited access to healthcare. Therefore, there is a need to create a low-cost preventative vaccine against the virus. Mature ZIKV particles contain a lipid bilayer, a positive sense single stranded RNA genome and three structural proteins: the envelope (E), membrane (M) and capsid (C) proteins. Congruently, to other members of the Flaviviridae family, ZIKV proteins are synthesized as a polyprotein precursor which needs to be processed to release the mature structural and non-structural viral proteins. Past studies have determined the ZIKV precursor protein is cleaved by a host furin protease which separates the Pr peptide and the M protein, while the host signal peptidase separates the M and E protein. Processing is important for correct folding of the E protein. In turn, the most important neutralizing antibodies upon infection are directed against epitopes of the E protein. In this work, we used a Bean Yellow Dwarf Viral vector system to transiently express, in Nicotiana benthamiana plants, a portion of the ZIKV polyprotein encoding the Pr, M and E proteins. I further demonstrate that plants can proteolytically process the polyprotein to yield the two integral membrane proteins M and E. These proteins can be shown to co-partition into a soluble membrane-particulate fraction, consistent with formation of enveloped virus-like particles (VLPs). This work provides the first step in creating a low-cost sustainable plant-based production system of ZIKV VLPs that can be explored as a potential component 0f a low-cost prophylactic vaccine against ZIKV.
ContributorsDi Palma, Michelle Pina (Author) / Mor, Tsafrir S (Thesis advisor) / Mason, Hugh S (Committee member) / Blattman, Joseph N (Committee member) / Arizona State University (Publisher)
Created2018
Description
In species with highly heteromorphic sex chromosomes, the degradation of one of the sex chromosomes can result in unequal gene expression between the sexes (e.g., between XX females and XY males) and between the sex chromosomes and the autosomes. Dosage compensation is a process whereby genes on the sex chromosomes achieve equal gene expression which prevents deleterious side effects from having too much or too little expression of genes on sex chromsomes. The green anole is part of a group of species that recently underwent an adaptive radiation. The green anole has XX/XY sex determination, but the content of the X chromosome and its evolution have not been described. Given its status as a model species, better understanding the green anole genome could reveal insights into other species. Genomic analyses are crucial for a comprehensive picture of sex chromosome differentiation and dosage compensation, in addition to understanding speciation.
In order to address this, multiple comparative genomics and bioinformatics analyses were conducted to elucidate patterns of evolution in the green anole and across multiple anole species. Comparative genomics analyses were used to infer additional X-linked loci in the green anole, RNAseq data from male and female samples were anayzed to quantify patterns of sex-biased gene expression across the genome, and the extent of dosage compensation on the anole X chromosome was characterized, providing evidence that the sex chromosomes in the green anole are dosage compensated.
In addition, X-linked genes have a lower ratio of nonsynonymous to synonymous substitution rates than the autosomes when compared to other Anolis species, and pairwise rates of evolution in genes across the anole genome were analyzed. To conduct this analysis a new pipeline was created for filtering alignments and performing batch calculations for whole genome coding sequences. This pipeline has been made publicly available.
In order to address this, multiple comparative genomics and bioinformatics analyses were conducted to elucidate patterns of evolution in the green anole and across multiple anole species. Comparative genomics analyses were used to infer additional X-linked loci in the green anole, RNAseq data from male and female samples were anayzed to quantify patterns of sex-biased gene expression across the genome, and the extent of dosage compensation on the anole X chromosome was characterized, providing evidence that the sex chromosomes in the green anole are dosage compensated.
In addition, X-linked genes have a lower ratio of nonsynonymous to synonymous substitution rates than the autosomes when compared to other Anolis species, and pairwise rates of evolution in genes across the anole genome were analyzed. To conduct this analysis a new pipeline was created for filtering alignments and performing batch calculations for whole genome coding sequences. This pipeline has been made publicly available.
ContributorsRupp, Shawn Michael (Author) / Wilson Sayres, Melissa A (Thesis advisor) / Kusumi, Kenro (Committee member) / DeNardo, Dale (Committee member) / Arizona State University (Publisher)
Created2016
Description
Satellite cells are adult muscle stem cells that activate, proliferate, and differentiate into myofibers upon muscle damage. Satellite cells can be cultured and manipulated in vitro, and thus represent an accessible model for studying skeletal muscle biology, and a potential source of autologous stem cells for regenerative medicine. This work summarizes efforts to further understanding of satellite cell biology, using novel model organisms, bioengineering, and molecular and cellular approaches. Lizards are evolutionarily the closest vertebrates to humans that regenerate entire appendages. An analysis of lizard myoprogenitor cell transcriptome determined they were most transcriptionally similar to mammalian satellite cells. Further examination showed that among genes with the highest level of expression in lizard satellite cells were an increased number of regulators of chondrogenesis. In micromass culture, lizard satellite cells formed nodules that expressed chondrogenic regulatory genes, thus demonstrating increased musculoskeletal plasticity. However, to exploit satellite cells for therapeutics, development of an ex vivo culture is necessary. This work investigates whether substrates composed of extracellular matrix (ECM) proteins, as either coatings or hydrogels, can support expansion of this population whilst maintaining their myogenic potency. Stiffer substrates are necessary for in vitro proliferation and differentiation of satellite cells, while the ECM composition was not significantly important. Additionally, satellite cells on hydrogels entered a quiescent state that could be reversed when the cells were subsequently cultured on Matrigel. Proliferation and gene expression data further indicated that C2C12 cells are not a good proxy for satellite cells. To further understand how different signaling pathways control satellite cell behavior, an investigation of the Notch inhibitor protein Numb was carried out. Numb deficient satellite cells fail to activate, proliferate and participate in muscle repair. Examination of Numb isoform expression in satellite cells and embryonic tissues revealed that while developing limb bud, neural tube, and heart express the long and short isoforms of NUMB, satellite cells predominantly express the short isoforms. A preliminary immunoprecipitation- proteomics experiment suggested that the roles of NUMB in satellite cells are related to cell cycle modulation, cytoskeleton dynamics, and regulation of transcription factors necessary for satellite cell function.
ContributorsPalade, Joanna (Author) / Wilson-Rawls, Norma (Thesis advisor) / Rawls, Jeffrey (Committee member) / Kusumi, Kenro (Committee member) / Newbern, Jason (Committee member) / Stabenfeldt, Sarah (Committee member) / Arizona State University (Publisher)
Created2020